1. Field of the Invention
The present invention relates to the field of cardiovascular and other diseases. More particularly, the present invention concerns compositions and methods of identification and use of isoform-selective activators or inhibitors of type 3 phosphodiesterase (PDE3). Other embodiments of the invention concern high-throughput screening for novel pharmaceuticals directed against PDE3 isoforms. In certain embodiments, the compositions and methods disclosed herein are of use for treatment of cardiomyopathy, pulmonary hypertension and related conditions.
2. Description of Related Art
PDE3 cyclic nucleotide phosphodiesterases hydrolyze cAMP and cGMP and thereby modulate cAMP- and cGMP-mediated signal transduction (Shakur et al., 2000a). These enzymes have a major role in the regulation of contraction and relaxation in cardiac and vascular myocytes. PDE3 inhibitors, which raise intracellular cAMP and cGMP content, have inotropic effects attributable to the activation of cAMP-dependent protein kinase (PK-A) in cardiac myocytes and vasodilatory effects attributable to the activation of cGMP-dependent protein kinase (PK-G) in vascular myocytes (Shakur et al., 2000a). When used in the treatment of dilated cardiomyopathy, PDE3 inhibitors such as milrinone, enoximone and aminone initially elicit favorable hemodynamic responses, but long-term administration increases mortality by up to 40% (Nony et al., 1994). This linkage of short-term benefits of PDE3 inhibition to deleterious effects on long-term survival in dilated cardiomyopathy is one of the most perplexing problems in cardiovascular therapeutics. However, it is thought that these biphasic effects reflect the compartmentally nonselective increases in intracellular cAMP content in cardiac myocytes current inhibitors display.
Clinical trials of the use of β-adrenergic receptor agonists, which, like PDE3 inhibitors, increase intracellular cAMP content in cardiac myocytes, were terminated prior to completion because of increased mortality in treated patients, while β-adrenergic receptor antagonists, which reduce intracellular cAMP content, have been shown to improve long-term survival despite initially adverse hemodynamic effects. These findings suggest that both the short-term benefits and long-term adverse effects of PDE3 inhibition are attributable to increases in intracellular cAMP content in cardiac myocytes (Movsesian, 1999).
The contradictory effects of nonspecific PDE3 antagonists may relate to the diverse intracellular processes regulated by cAMP in cardiac and vascular cells. Upon activation by cAMP, PK-A phosphorylates dozens of proteins in separate intracellular compartments that are involved in contraction and relaxation, glycogen metabolism, gene transcription, intracellular Ca2+ cycling and signal autoregulation. Phosphorylation of cAMP-response element-binding protein (CREB), for example, activates the transcription of genes containing cAMP response elements (Shaywitz and Greenberg, 1999). Transgenic mice expressing a dominant non-phosphorylatable CREB in cardiac myocytes develop a dilated cardiomyopathy that very closely resembles the human disease (Fentzke et al., 1998), suggesting that CREB phosphorylation may be desirable in dilated cardiomyopathy.
Another example of cAMP effects is the phosphorylation of phospholamban, which relieves its inhibition of SERCA2, the Ca2+-transporting ATPase of the sarcoplasmic reticulum (Simmerman and Jones, 1998). Ablation of phospholamban in muscle LIM protein (MLP)−/− mice with dilated cardiomyopathy results in the restoration of normal chamber size and contractility (Minamisawa et al., 1999), suggesting that phospholamban phosphorylation may also be beneficial in cardiomyopathy.
Other substrates phosphorylated by PK-A may contribute to adverse effects on long-term survival. Phosphorylation of L-type Ca2+ channels increases their open probability and may be arrhythmogenic (Fischmeister and Hartzell, 1990), while phosphorylation of proteins in the mitogen-activated protein kinase (MAP kinase) cascade may alter myocardial gene transcription so as to speed the progression of the disease (Cook and McCormick, 1993; Lazou et al., 1994).
Raising cAMP content in cardiac myocytes via mechanisms such as activation of β1-adrenergic, β2-adrenergic or prostaglandin receptors or non-selective phosphodiesterase inhibition by isobutylmethylxanthine, affects cAMP content differentially in intracellular compartments represented in cytosolic and microsomal fractions of cardiac muscle, resulting in different patterns of protein phosphorylation and different physiologic responses (Hayes et al., 1980; Xiao and Lakatta, 1993; Xiao et al., 1994; Rapundalo et al., 1989; Jurevicius and Fischmeister, 1996). These considerations are particularly relevant to the pathophysiology of dilated cardiomyopathy, in which receptor-mediated and receptor-independent reductions in cAMP generation are prominent features (Movsesian, 1999; Lutz, et al., 2001). Comparison of cytosolic cAMP content in cytosolic and microsomal fractions between failing and non-failing hearts shows greater reduction in cAMP content in microsomal fractions of failing myocardium than in cytosolic fractions (Bohm, 1994).
The phosphorylation of individual substrates of PK-A may be differentially regulated in response to extracellular signals. Evidence for differential regulation comes from experiments examining the effects of stimulating adenylate cyclase activity and cAMP formation via β1-adrenergic, β2-adrenergic or PGE1 receptors. Activation of β-adrenergic receptors increases cAMP content in both cytosolic and microsomal fractions of cardiac myocytes and elicits contractile responses, while activation of PGE1 receptors increases cytosolic but not microsomal cAMP content and evokes no contractile response (Hayes et al., 1980; Buxton and Brunton, 1983). Increases in the amplitude of intracellular Ca2+ transients in response to β1-adrenergic receptor activation correlate with changes in microsomal cAMP content and are accompanied by increases in phospholamban phosphorylation. Conversely, activation of β2-adrenergic receptors results in an increase in the amplitude of intracellular Ca2+ transients that does not correlate with changes in microsomal cAMP content and occurs without increases in phospholamban phosphorylation (Hohl and Li, 1991; Xiao et al., 1993, 1994). Thus, activation of different receptors linked to cAMP metabolism can elicit different responses in cardiac tissues.
β-adrenergic receptor stimulation and nonselective phosphodiesterase inhibition have different effects on cAMP-activated protein phosphorylation in cardiac myocytes (Rapundalo et al., 1989; Jurvicius and Fischmeister, 1996) that are relevant to the pathophysiology of dilated cardiomyopathy. In that condition, a down-regulation of β1-adrenergic receptors and an uncoupling of β-adrenergic receptor occupancy and adenylate cyclase stimulation (attributable to increases in β-adrenergic receptor kinase, Gαi and nucleoside diphosphate kinase) contribute to an impairment in cAMP generation (Movsesian, 1999; Lutz et al., 2001). Studies of cAMP content in cytosolic and microsomal fractions of failing and non-failing hearts demonstrate a far greater reduction in cAMP content in microsomal fractions than in cytosolic fractions of failing myocardium (Bohm et al., 1994). Taken together, these results indicate that cAMP content in different intracellular compartments can be selectively regulated to invoke different responses reflecting the phosphorylation of different substrates of PK-A. Further, this regulation is altered in dilated cardiomyopathy.
Different isoforms of PDE3 are expressed in cardiac and vascular myocytes and are localized to different intracellular compartments. The different PDE3 isoforms may differ in their regulation by PK-A and PK-B (protein kinase B, also known as Akt). PK-B, a downstream effector of insulin-like growth factors, is an anti-apoptotic mediator in cardiac myocytes (Fujio et al., 2000; Matsui et al., 1999; Wu et al., 2000). PK-B may also be involved in proliferative responses in vascular myocytes (Rocic and Lucchesi, 2001; Duan et al., 2000; Sandirasegarane et al., 2000). These findings suggest that different PDE3 isoforms may be involved in cell- and compartment-selective responses to different signals that have been implicated in the pathophysiology of dilated cardiomyopathy and/or pulmonary hypertension. Different PDE3 isoforms in cardiac and vascular myocytes may regulate functionally distinct pools of cAMP and cGMP involved in the phosphorylation of different substrates of PK-A and PK-G, and these isoforms may be regulated in response to different extracellular signals.
Until the present invention, it was not possible to develop isoform-selective inhibitors or activators of PDE3 to use in the treatment of cardiomyopathy and/or pulmonary hypertension. Isoform-selective PDE3 inhibitors may provide a beneficial effect on cardiac output without the long-term mortality associated with non-specific PDE3 inhibitors. Isoform-selective PDE3 activators may have beneficial anti-apoptotic effects in patients with dilated cardiomyopathy and/or pulmonary hypertension whose hemodynamic status is not too compromised to tolerate a reduction in cardiac contractility, without concomitant arrhythmogenic effects attributable to increases in cytosolic cAMP content. A paradigm for the latter is the use of β-adrenergic receptor antagonists in the treatment of dilated cardiomyopathy.